Tissue inhibitor of metalloproteinases, hereinafter referred to as TIMP, a sialoglycoprotein having a molecular weight of about 30,000, is a specific inhibitor of the principal matrix metalloproteinases, i.e., interstitial collagenase, gelatinase/type IV collagenase (72 kDa and 92 kDa) and stromelysin. It, however, is known to have no inhibitory action on bacterial collagenase and thermolysin although they are likewise metalloenzymes (Cawston, In Proteinase Inhibitors (eds. Barrett A. J. and Salvesen G.) Elsevier, Amsterdam, pp. 589-610, 1986). TIMP is synthesized by a variety of cells, i.e., fibroblast cells, epithelial cells, endothelial cells, osteoblast cells, chondrocytes, platelets, macrophages and some tumor cells, and is also found in all kinds of mammalian body fluids (Welgus and Stricklin, J. Biol. Chem., 258, 12259-12264, 1983; Kodama et al., Matrix 9, 1-6, 1989). All this suggests that TIMP is a basic and universal protein in mammals.
Docherty et al. (Nature 318, 66-69, 1985) established the total amino acid sequence of TIMP on the basis of cDNA analysis. This sequence has been found to be identical in its entirety with the amino acid sequence of erythroid potentiating activity (EPA) produced by HTLV-II infected Mo T lymphoblasts (Gasson et al., Nature, 315, 768-771, 1985). EPA is also known to accelerate in vitro the growth of the human erythroblastic leukemia cell line K562 (Avalos et al., Blood, 71, 1720-1725, 1988). Recently, Hayakawa et al. (FEBS Lett., 268, 125-128, 1990) have demonstrated that TIMP produced by KM 102, a human bone marrow interstitial cell line, accelerates the colony formation of burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) cells. On the other hand, it has been demonstrated that the 22 kDa protein which is an expression product of 16C8, one of the "cell division cycle" genes of normal mouse fibroblast cells, is entirely identical with mouse TIMP (Gewert et al., EMBO J., 6, 651-657, 1987), and corresponds to human TIMP/EPA (Edwards et al., Nucleic Acid Res., 14, 8863-8878, 1986). These facts are suggestive of the possibility of TIMP being active as a cell growth factor not only for erythroid precursor (stem) cells but also for other cells.
The present inventors previously succeeded in the preparation of mouse monoclonal antibodies to bovine dental pulp TIMP, and found that antibodies from several out of these clones react specifically with TIMP present in human dental pulp tissue cultures (Kodama et al., Collagen Rel. Res. 7, 341-350, 1987). The present inventors also succeeded in determining with high sensitivity TIMP in the bovine or human body fluid or blood by sandwich enzyme immunoassay (EIA) using combinations of these antibodies (Kodama et al., Matrix 9, 1-6, 1989). It has been well known in the art that the addition of fetal calf serum (FCS) to tissue culture media results in acceleration of cell growth without exception, but it remains to be established what component (or components) in FCS acts (or act) as a growth factor.
The present inventors found the TIMP concentration in FCS to be about 250 ng/ml when measured by a sandwich EIA using the above mentioned monoclonal antibodies. The monoclonal antibodies were likewise used to prepare an affinity column for removal of TIMP from FCS, and a culture medium prepared from the thus obtained TIMP-free FCS was used to culture human gingival fibroblast cells. Such human gingival fibroblast cells cultured in TIMP-free FCS medium showed little growth up to day 3, and upon further addition to the culture medium of anti-TIMP monoclonal antibodies, no growth. On the other hand, when such culture was carried out in a culture medium prepared from TIMP-free FCS while adding purified TIMP thereto, it was found that the cell growth was commensurate with the concentration of TIMP added and, at a TIMP concentration of 100 ng/ml, the cell growth reached a level comparable to that as found in conventional 10% FCS-supplemented culture media.
It was also found that when adherent and nonadherent cells were cultured in serum-free medium supplemented with 100 ng/ml of TIMP a cell growth acceleration effect comparable to that as found with conventional 10% FCS-supplemented culture media was achieved.
On the basis of these findings, the present inventors have confirmed the effect of TIMP as a growth factor on cell growth, and succeeded in providing serum-free media extremely suitable for the culture of adherent and nonadherent cells by adding TIMP as a substitute for FCS to serum-free media.